Botulinum Neurotoxin A Receptor and the Use Thereof

ABSTRACT

The present invention is based on the identification of synaptic vessel glycoprotein SV2 as the BoNT/A receptor and the further identification of various BoNT/A-binding fragments of SV2. The disclosure here provides new tools for diagnosing and treating botulism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/546,880, filed Oct. 12, 2006, which claims the benefit of U.S.Provisional Application No. 60/726,879, filed on Oct. 14, 2005, both ofwhich are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.AI057153 and AI057744 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Botulinum neurotoxin A (BoNT/A) is one of seven botulinum neurotoxins(designated BoNT/A-G) produced by the anaerobic bacteria strainClostridium botulinum (Schiavo G et al., Physiol. Rev. 80:717-766,2000). BoNTs block neurotransmitter release by cleaving members of themembrane fusion machinery composed of SNAP-25, vamp-2/synaptobrevin(Syb), and syntaxin (Jahn R and Niemann H, Ann. NY Acad. Sci.733:245-255, 1994; Schiavo G. et al., supra, 2000). Cleavage of theseproteins in motor nerve terminals blocks acetylcholine release at theneuromuscular junction (NMJ) which causes paralysis and may lead todeath due to respiratory failure (Schiavo G. et al., supra, 2000;Simpson L L, Ann. Rev. Pharmacol. Toxicol. 44:167-193, 2004). Due toextreme potency and lethality as well as ease of use and transport,BoNTs are considered one of the six most dangerous potentialbioterrorism threats (designated by Center for Disease Control of UnitedStates) (Arnon S. et al., JAMA 285:1059-1070, 2001). According to theAmerican Medical Society, as little as one gram of crystalline toxin issufficient to kill one million people.

Currently, the standard test for BoNTs is the mouse bioassay availableat the Centers for Disease Control and Prevention (CDC) and selectlaboratories across the country. The test involves treating mice withclinical samples suspected of carrying one of the BoNTs. The mice areimmunized against the various BoNTs, and only those mice immunizedagainst the specific BoNT present in the sample will survive. Althoughthe test is sensitive in that it can detect as little as 0.03 ng of aBoNT, it is expensive and takes days to complete. On the treatment side,equine antitoxin containing antibodies against a BoNT is the therapy ofchoice and its effectiveness depends on timely treatment. Thistreatment, however, has all the disadvantages of a horse serum productsuch as the risks of anaphylaxis and serum sickness. Many times,treatment begins before botulism is confirmed as the diagnostic testtakes days which is too long to wait for effective treatment. Therefore,there is a need in the art for alternative detection and treatmentstrategies.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the identification of synaptic vesselglycoprotein SV2 as the BoNT/A receptor and the further identificationof various BoNT/A-binding fragments of SV2. The disclosure here providesnew tools for diagnosing and treating botulism.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows that stimulating synaptic vesicle exocytosis increasesBoNT/A binding to diaphragm motor nerve terminals and culturedhippocampal neurons. Panel a: Mouse hemi-diaphragm preparations wereexposed to BoNT/A (25 nM) in either resting conditions (control buffer)or stimulated conditions (High K⁺ buffer: 45 mM KCl). The tissue wasfixed and permeabilized. Neuromuscular junctions (NMJs) were labeledwith Alexa-488 conjugated α-BTX. BoNT/A was detected with a polyclonalBoNT/A antibody and a Cy3-conjugated secondary antibody. The overlay ofregions indicated by rectangles was enlarged to show that BoNT/Astaining mirrors α-BTX staining at individual NMJs. Right panel: BoNT/Abinding to NMJs was quantified. NMJ regions (region of interest, ROI)were defined by α-BTX signals. The intensity of BoNT/A staining wasnormalized to α-BTX signals and the ratios were plotted on the Y-axis.Stimulation with high K⁺ buffer resulted in an approximate 6-foldincrease in BoNT/A intensity. Error bars represent SEM (n=27-28 images).Panel b: Cultured rat hippocampal neurons were exposed to BoNT/A (10 nM)and an antibody against the luminal domain of synaptotagmin I (Syt I_(N)Ab; CI604.4, 1:40) for 1 min in three different buffer conditions: (1)Normal buffer (PBS), (2) High K⁺ (56 mM KCl), and (3) High K⁺/No Ca²⁺(without Ca²⁺). Stimulating neurons with high K⁺increased Syt I_(N) Abimmunofluorescence signals. This increase was not seen withoutextracellular Ca²⁺. BoNT/A signals largely co-localize with Syt I_(N) Absignals. The third image column on the right shows images obtained fromdifferential interference contrast microscopy (DIC). Panel c: Rathippocampal neurons pretreated with BoNT/B (30 nM, 24 h) were exposed toBoNT/A (10 nM) for 10 min in High K⁺ buffer. Cells were fixed andimmunostained for Syb II and BoNT/A. BoNT/A binding is abolished byBoNT/B treatment, which cleaves Syb II in neurons. Neurons that were nottreated with BoNT/B served as controls. The third image column on theright shows images obtained from DIC microscopy.

FIG. 2 shows that BoNT/A binds directly to SV2 luminal domains. Panel a:Monoclonal antibodies against the synaptic vesicle proteinssynaptophysin (Syp, CI7.2) and SV2 (pan-SV2), were used toco-immunoprecipitate BoNT/A (100 nM) from rat brain detergent extract.Control samples without antibodies (No Ab) were carried out in parallel.Immunoprecipitated toxin and vesicle proteins were detected by westernblot. BoNT/A co-immunoprecipitated with SV2, but not Syp. Panel b:Co-immunoprecipitations of BoNT/A from mouse brain detergent extractwere carried out with pan-SV2 antibodies, with or without exogenousgangliosides (mixture of bovine brain gangliosides, 0.6 mg/ml), at theindicated BoNT/A concentrations. Adding gangliosides increased theamount of BoNT/A co-immunoprecipitated by SV2 antibodies. Panel c: The4^(th) luminal domains of all three SV2 isoforms (see panel d for SV2topology) were purified as GST-tagged proteins and immobilized onglutathione-sepharose beads. Pull down assay were carried out using 8 μgimmobilized proteins and 100 nM toxins (BoNT/A, B or E). Bound materialswere analyzed by western blot with anti-BoNT/A, B, E antibodies. BoNT/Abinds directly to all SV2 isoforms with the luminal domain of SV2Cshowing the highest apparent affinity. Panel d: Schematic view ofputative SV2 topology. Each circle represents a residue. Filled circlesindicate conserved residues in all SV2A, B and C isoforms, gray circlesare residues conserved in two of SV2 isoforms, and open circlesrepresent non-conserved residues. SV2 contains 12 transmembrane domainswith its N- and C-terminus facing the cytoplasm. The 4^(th) luminaldomain (L4) lies inside vesicles and contains three putativeN-glycosylation sites, indicated. The critical region in SV2C for BoNT/Abinding is indicated by arrows (see panel e for details). Panel e: Aseries of truncation mutants within SV2C-L4 were generated as GST fusionproteins and tested for BoNT/A binding. Binding assays were performed asdescribed in panel b and analyzed by western blot. The critical regionfor BoNT/A binding was mapped to a short fragment (residues 529-566 inSV2C), which alone maintains the ability to bind BoNT/A. Immobilized GSTfusion proteins were shown by Ponceau S staining to ensure that equalamount of immobilized protein were used in the assay. The proteinsequence of this region is aligned with regions of SV2A and B (all arerat sequences), with putative N-glycosylation sites indicated byasterisks.

FIG. 3 shows the block of BoNT/A binding and entry into hippocampalneurons and motor nerve terminals by an SV2C luminal fragment. Panel a,left panel: hippocampal neurons were exposed to BoNT/A (10 nM) and SytI_(N) Ab in High K⁺ buffers for 10 min, with the presence of eithercontrol protein (soluble GST, 10 μM) or SV2C-L4 (soluble GST taggedSV2C-L4 fragment, 10 μM). Cells were washed and fixed. Binding anduptake of Syt I_(N) Ab and BoNT/A were analyzed through subsequentimmunostaining SV2C-L4 did not affect Syt I_(N) Ab uptake into neurons,but reduced BoNT/A binding to the same neurons. Panel a, right panel:The experiment was carried out as described above except using BoNT/Binstead of BoNT/A. SV2C-L4 did not affect BoNT/B binding to neurons.Panel b: Hippocampal neurons were incubated with BoNT/A (10 nM), in thepresence of either GST proteins or SV2C-L4, for 10 min in High K⁺buffers. Cells were washed three times and further incubated for 6 hrsin culture medium. Cells were then fixed and permeabilized. Cleavage ofSNAP-25 was detected using a monoclonal antibody (anti-SNAP-25-C) thatonly recognizes cleaved SNAP-25 (but not intact full-length SNAP-25).SV2C-L4 prevented the cleavage of native SNAP-25 by BoNT/A. The thirdimage column on the right shows images obtained from DIC microscopy.Panel c: Mouse hemi-diaphragm preparations were exposed to BoNT/A (10nM) or BoNT/B (10 nM) in the presence of either GST protein or SV2C-L4for 30 min in High K⁺ buffer. Tissues were washed, fixed andpermeabilized. NMJs were labeled with α-BTX. BoNT/A and B were detectedwith their polyclonal antibodies, respectively. SV2C-L4 specificallyreduced binding of BoNT/A to NMJs, while it has no effect on BoNT/Bbinding. Panel d: Binding of BoNT/A and B to NMJs, based on imagescollected in panel c, were quantified as described in FIG. 1 a. SV2C-L4significantly reduced BoNT/A binding (65% reduction compared to control,P<0.0001, t-test, n=76-90 images), but did not affect BoNT/B binding(P>0.05, t-test, n=49-55 images). Error bars represent SEM.

FIG. 4 shows that BoNT/A binding is abolished in SV2A and B knockouthippocampal neurons. Panel a: Hippocampal neurons from SV2B knockout(SV2B(−/−)) mice and wild-type (WT) littermate controls were cultured.Neurons were exposed to BoNT/A (15 nM) and BoNT/B (7.5 nM) in High K⁺for 10 min. They were washed three times to reduce surface bound toxins,fixed and permeabilized Immunofluorescence signals for BoNT/A and B weredetected and quantified, and plotted as normalized intensity ratios (%WT signals). SV2B(−/−) neurons displayed significantly reduced BoNT/Auptake (28% reduction compared to WT, P<0.0001, t-test, n=18 images).BoNT/B uptake level remained the same for SV2B(−/−) and WT neurons(P>0.05, t-test, n=22 images). Error bars represent SD. Panel b:Hippocampal neurons from littermates with the following genotypes:SV2A(+/+)SV2B(−/−), SV2A(+/+)SV2B(−/−), and double knockoutSV2A(−/−)SV2B(−/−) were cultured. Cultures were exposed to BoNT/A (10nM) and BoNT/B (7.5 nM) simultaneously for 10 min. Cells were washedthree times, fixed and permeabilized. Triple immunostaining wasperformed (BoNT/B: rabbit anti-BoNT/B; BoNT/A: human anti-BoNT/A; SV2:mouse pan-SV2). Binding of BoNT/B to neurons was not altered betweendifferent genotypes. SV2B knockout and SV2A heterozygotes(SV2A(+/−)SV2B(−/−)) showed reduced BoNT/A binding. SV2A/B doubleknockouts showed no binding of BoNT/A. Panel c: Images collected inpanel b were thresholded to only include neurons. The average intensity(background subtracted) was plotted as normalized data (% of SV2A(+/+)).SV2A(+/−)SV2B(−/−) neurons displayed a 53% reduction compared toSV2A(+/+)SV2B(−/−), and SV2A/B double knockouts showed no binding ofBoNT/A. The binding of BoNT/B remained the same for all genotypes(P>0.05, t-test, n=11 images).

FIG. 5 shows that introducing SV2A, B or C in SV2A/B double knockoutneurons rescues BoNT/A binding. Rat SV2A, B and C were subcloned into alentiviral vector under control of a neuronal specific synapsinpromoter, and were used to transfect hippocampal neurons from SV2A/Bdouble knockout mice. Forty-eight hrs after transfection, neurons wereexposed to BoNT/A (10 nM) for 10 min. Cells were washed three times,fixed, and permeabilized Immunostaining for SV2 and BoNT/A wereperformed as described in FIG. 4 b. Transfected neurons were identifiedby GFP expression, which is under control of a separated synapsinpromoter in the vector. Expression of exogenous un-tagged SV2 isoformswere confirmed by SV2 staining, and BoNT/A selectively bound totransfected cells. The overlay images of regions indicated by whiterectangles are enlarged to show the high degree of colocalizationbetween SV2 expression and BoNT/A signals.

FIG. 6 shows that SV2 knockout mice have reduced BoNT/A binding atdiaphragm motor nerve terminals and are more resistant to BoNT/A. Panela: Mouse diaphragms were prepared from wild-type (WT) andSV2A(+/−)SV2B(−/−) mice. Half of the diaphragm was exposed to BoNT/A (25nM) in stimulated conditions for 1 hr. The tissue was fixed andimmunostained with α-BTX and BoNT/A antibody as described in FIG. 1 a.The other half of the diaphragm was exposed to BoNT/B (25 nM), andimmunostained in parallel. Representative images are shown. Panel b:Images collected in panel a were quantified as described in FIG. 1 a.SV2A(+/−)SV2B(−/−) mice showed significantly less BoNT/A bindingcompared to WT (72% reduction, P<0.001, t-test, n=47 images), whileBoNT/B binding is the same (P>0.05, t-test, n=20 images). Panel c: Thesusceptibility of SV2B(−/−) mice and their WT littermates to BoNT/A wasdetermined by a rapid time-to-death assay. The same amount of BoNT/A wasinjected into each mouse, and their survival time (time-to-death)recorded. The average effective toxicity (LD₅₀/ml) were estimated fromtime-to-death data using a standard curve. SV2B(−/−) mice livesignificantly longer than WT mice (43 min versus 33 min, P<0.05, pairedt-test). The effective toxicity of injected BoNT/A in WT mice is about2.5-fold greater than SV2B knockout mice.

FIG. 7 shows that a peptide containing the BoNT/B binding sitespecifically inhibits BoNT/B, but not BoNT/A binding to hippocampalneurons, and SV2C-L4 does not affect BoNT/E binding. Panel a: PeptideP21 is derived from the synaptotagmin II luminal domain (Dong M et al.,J. Cell. Biol. 162:1293-1303, 2003). P21S is a scrambled version of P21that serves as a control (Dong M et al., supra, 2003). Culturedhippocampal neurons were exposed to BoNT/B (10 nM) and Syt I_(N) Ab inHigh K⁺ buffers for 10 min, in the presence of P21 (30 μM) or P21S.Cells were washed and fixed. Binding and uptake of Syt I_(N) Ab andBoNT/B were analyzed through subsequent immunostaining as described inFIG. 3 a. P21 inhibited BoNT/B binding to neurons, while uptake of SytI_(N) Ab is not affected. Panel b: Experiments were carried out asdescribed in panel a with BoNT/A instead of BoNT/B. P21 peptide did notaffect BoNT/A binding to hippocampal neurons. Panel c: Hippocampalneurons were exposed to BoNT/E (10 nM) and Syt I_(N) Ab in High K⁺buffers for 10 min, in the presence of GST (10 μM) or SV2C-L4 (10 μM).Binding of BoNT/E was detected with a polyclonal anti-BoNT/E antibody.SV2C-L4 did not affect BoNT/E binding to neurons.

FIG. 8 shows that motor nerve terminals at mouse diaphragm express SV2A,B and C. Mouse hemi-diaphragms were excised, immediately fixed in 4%paraformaldehyde for 30 min, permeabilized, and blocked. Expression ofSV2A, B or C was detected by their specific polyclonal antibodies(1:1000). NMJs were labeled with α-BTX. All SV2 isoforms were observedat NMJs, presumably at presynaptic nerve terminals.

FIG. 9 shows an alignment of partial sequences of rat SV2C (SEQ IDNO:6), human SV2A (SEQ ID NO:14), human SV2B (SEQ ID NO:16), human SV2C(SEQ ID NO:18), mouse SV2A (SEQ ID NO:8), mouse SV2B (SEQ ID NO:10), andmouse SV2C (SEQ ID NO:12).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of synaptic vesselglycoprotein SV2 as the BoNT/A receptor as well as the identification ofvarious BoNT/A-binding fragments of SV2. The disclosure here providesnew prevention and treatment strategies for BoNT/A toxicity and thebotulism disease. The disclosure here also provides new tools foridentifying agents that can reduce SV2-BoNT/A binding, BoNT/A cellularentry, and BoNT/A toxicity.

In some species (e.g., human, rat, and mouse), three SV2 isoforms,namely SV2A, SV2B, and SV2C, have been identified. Using rat SV2 as anexample, the inventors found that all three isoforms are capable ofbinding to and serve as the receptor for BoNT/A. In other species suchas bovine and electric ray (Discopyge ommatta), only one isoform hasbeen identified so far. The bovine SV2 cDNA is closer to SV2A than SV2Band SV2C, and the electric ray SV2 cDNA is closer to SV2C than SV2A andSV2B. It is known in the art that the function and amino acid sequencesof SV2A, SV2B, and SV2C are conserved across animal species (mammalianspecies in particular). At protein level, there is at least 62% identityamong known SV2 proteins (human, mouse, rat, bovine, and electric ray)and at least 57% identity among the luminal domains of known SV2proteins. For known SV2A and bovine SV proteins, the amino acid sequenceidentity is over 98% for the whole protein and 100% for the luminaldomain. For known SV2B proteins, the amino acid sequence identity isover 94% for the whole protein and over 96% for the luminal domain. Forknown SV2C and electric ray SV proteins, the amino acid sequenceidentity is over 79% (over 96% for mammalian species) for the wholeprotein and over 76% (over 97% for mammalian species) for the luminaldomain. The amino acid sequence identity among rat SV2A, B, and Cluminal domains is 76% and the amino acid sequence identity among mouseSV2A, B, and C luminal domains is 75%. Although the disclosure here isbased on the discovery made with rat SV2A, SV2B, and SV2C, it applies toall animal species including all mammalian species. For example, whilecertain rat SV2C fragments have been shown to be capable of binding toBoNT/A, corresponding fragments from rat SV2A, rat SV2B as well ascorresponding fragments from other SV2 homologs are expected to becapable of binding to BoNT/A. Corresponding domains and fragments amongall SV2 proteins can be identified using any alignment program familiarto a skilled artisan. For example, the GCG software from Accelrys (SanDiego, Calif.) can be used for this purpose (e.g., the MegaAlign programwith default parameters).

An SV2 protein typically contains 12 transmembrane domains, 7cytoplasmic domains, and one large luminal domain (luminal domain 4, L4)(Janz R and Sudhof T C, Neuroscience 94:1279-1290, 1999). In the case ofrat SV2A, SV2B, and SV2C, the luminal domain spans from amino acid 468to amino acid 595, amino acid 411 to amino acid 536, and amino acid 454to amino acid 580, respectively. The inventors have determined thatBoNT/A binds to an SV2 protein at its luminal domain. In particular, theinventors have demonstrated that rat SV2C luminal domain fragments aminoacids 529-562 and amino acids 454-546 and various other fragmentscontaining the above fragments are capable of binding to BoNT/A.Fragment amino acids 529-566 binds almost as efficiently as the luminaldomain itself Fragments shorter than that spanning amino acids 529-562or 454-546 may also be able to bind to BoNT/A and a skilled artisan canreadily identify these fragments by routine truncation experiments.

Furthermore, a peptide that is at least 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% identical to any of the BoNT/A-binding fragments of an SV2protein discussed above and any such binding fragments with one or moreconservative substitutions are expected to be able to bind to BoNT/A. Itis well known in the art that the amino acids within the sameconservative group can typically substitute for one another withoutsubstantially affecting the function of a protein. For the purpose ofthe present invention, such conservative groups are set forth in Table 1based on shared properties.

TABLE 1 Conservative substitution. Original Residue ConservativeSubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr(Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

The cDNA and amino acid sequences for rat SV2A (cDNA sequence is setforth in SEQ ID NO:1 and amino acid sequence is set forth in SEQ IDNO:2), SV2B (cDNA sequence is set forth in SEQ ID NO:3 and amino acidsequence is set forth in SEQ ID NO:4), and SV2C (cDNA sequence is setforth in SEQ ID NO:5 and amino acid sequence is set forth in SEQ IDNO:6) can be found at GenBank Accession Nos. NM_(—)057210, L10362, andNM_(—)031593, respectively. The cDNA and amino acid sequences for mouseSV2A (cDNA sequence is set forth in SEQ ID NO:7 and amino acid sequenceis set forth in SEQ ID NO:8), SV2B (cDNA sequence is set forth in SEQ IDNO:9 and amino acid sequence is set forth in SEQ ID NO:10), and SV2C(cDNA sequence is set forth in SEQ ID NO:11 and amino acid sequence isset forth in SEQ ID NO:12) can be found at GenBank Accession Nos.NM_(—)022030, NM_(—)153579, and XM_(—)991257, respectively. The cDNA andamino acid sequences for human SV2A (cDNA sequence is set forth in SEQID NO:13 and amino acid sequence is set forth in SEQ ID NO:14), SV2B(cDNA sequence is set forth in SEQ ID NO:15 and amino acid sequence isset forth in SEQ ID NO:16), and SV2C (cDNA sequence is set forth in SEQID NO:17 and amino acid sequence is set forth in SEQ ID NO:18) can befound at GenBank Accession Nos. NM_(—)014849, BCO30011, and BC100827,respectively. The cDNA and amino acid sequences for bovine SV2 (cDNAsequence is set forth in SEQ ID NO:19 and amino acid sequence is setforth in SEQ ID NO:20) can be found at GenBank Accession No.NM_(—)173962. The cDNA and amino acid sequences for electric ray(Discopyge ommatta) SV2 (cDNA sequence is set forth in SEQ ID NO:21 andamino acid sequence is set forth in SEQ ID NO:22) can be found atGenBank Accession No. L23403.

Polypeptides, Nucleic Acids, Vectors, and Host Cells

The term “isolated polypeptide” or “isolated nucleic acid” used hereinmeans a polypeptide or nucleic acid isolated from its naturalenvironment or prepared using synthetic methods such as those known toone of ordinary skill in the art. Complete purification is not requiredin either case. The polypeptides and nucleic acids of the invention canbe isolated and purified from normally associated material inconventional ways such that in the purified preparation the polypeptideor nucleic acid is the predominant species in the preparation. At thevery least, the degree of purification is such that the extraneousmaterial in the preparation does not interfere with use of thepolypeptide or nucleic acid of the invention in the manner disclosedherein. The polypeptide or nucleic acid is preferably at least about 85%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure.

Further, an isolated nucleic acid has a structure that is not identicalto that of any naturally occurring nucleic acid or to that of anyfragment of a naturally occurring genomic nucleic acid spanning morethan one gene. An isolated nucleic acid also includes, withoutlimitation, (a) a nucleic acid having a sequence of a naturallyoccurring genomic or extrachromosomal nucleic acid molecule but which isnot flanked by the coding sequences that flank the sequence in itsnatural position; (b) a nucleic acid incorporated into a vector or intoa prokaryote or eukaryote genome such that the resulting molecule is notidentical to any naturally occurring vector or genomic DNA; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), or a restriction fragment;and (d) a recombinant nucleotide sequence that is part of a hybrid gene,i.e., a gene encoding a fusion protein. Specifically excluded from thisdefinition are nucleic acids present in mixtures of clones, e.g., asthese occur in a DNA library such as a cDNA or genomic DNA library. Anisolated nucleic acid can be modified or unmodified DNA or RNA, whetherfully or partially single-stranded or double-stranded or eventriple-stranded. A nucleic acid can be chemically or enzymaticallymodified and can include so-called non-standard bases such as inosine.

As used in this application, “percent identity” between amino acid ornucleotide sequences is synonymous with “percent homology,” which can bedetermined using the algorithm of Karlin and Altschul (Proc. Natl. Acad.Sci. USA 87, 2264-2268, 1990), modified by Karlin and Altschul (Proc.Natl. Acad. Sci. USA 90, 5873-5877, 1993), or other methods familiar toa skilled artisan. The noted algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (J. Mol. Biol. 215, 403-410,1990). BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a polynucleotide of interest. BLAST protein searches canbe performed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to a reference polypeptide. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (Nucleic Acids Res. 25, 3389-3402,1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. An example of another program for aligning two amino acidsequences (MegaAlign, GCG) is provided earlier in the specification.

In one aspect, the present invention relates to an isolated polypeptidecontaining an amino acid sequence that is at least 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% identical to that of a BoNT/A-binding fragment ofan SV2 protein over the entire length of the binding fragment or anamino acid sequence of a BoNT/A-binding fragment of an SV2 protein withone or more conservative substitutions. Preferably, the above isolatedpolypeptide is capable of binding to BoNT/A. Specifically excluded fromthe polypeptide of the present invention is one that contains a fulllength SV2 protein. In one embodiment, an isolated polypeptide thatconsists of an SV2 luminal domain or that contains an SV2 luminal domainwherein the domain is flanked at one or both ends by a non-nativeflanking amino acid sequence is also excluded from the presentinvention. Examples of BoNT/A binding fragments of SV2 proteins includebut are not limited to (i) amino acids 529-562 of rat SV2C, (ii) aminoacids 486 to 519 of rat SV2B, (iii) amino acids 543 to 576 of rat SV2A,(iv) a fragment of a homolog of the rat SV2C, SV2B, or SV2A wherein thefragment corresponds to amino acids 529-562 of rat SV2C, amino acids 486to 519 of rat SV2B, or amino acids 543 to 576 of rat SV2A, respectively(see FIG. 9 for examples), (v) amino acids 454-546 of rat SV2C, (vi)amino acids 411 to 503 of rat SV2B, (vii) amino acids 468 to 560 of ratSV2A, and (viii) a fragment of a homolog of the rat SV2C, SV2B, or SV2Awherein the fragment corresponds to amino acids 454-546 of rat SV2C,amino acids 411 to 503 of rat SV2B, or amino acids 468 to 560 of ratSV2A, respectively (see FIG. 9 for examples).

Preferred BoNT/A binding fragments of SV2 proteins include but are notlimited to (i) amino acids 529-566 of rat SV2C, (ii) amino acids 486 to523 of rat SV2B, (iii) amino acids 543 to 580 of rat SV2A, (iv) afragment of a homolog of the rat SV2C, SV2B, or SV2A wherein thefragment corresponds to amino acids 529-566 of rat SV2C, amino acids 486to 523 of rat SV2B, or amino acids 543 to 580 of rat SV2A, respectively.Other preferred BoNT/A binding fragments include the luminal domains ofSV2 proteins.

In one embodiment, the polypeptide of the present invention is about thesize of an SV2 luminal domain or shorter. For example, the polypeptideof the present invention can be shorter than 129, 128, 127, or 126 aminoacids. In another embodiment, the polypeptide of the present inventionis shorter than 125, 120, 110, 100, 90, 80, 70, 60, 50, or 40 aminoacids.

In another embodiment, the polypeptide of the present invention issoluble in an aqueous solvent (e.g., water with or without otheradditives). By soluble in an aqueous solvent, we mean that thepolypeptide exhibits a solubility of at least 10 μg/ml, preferably atleast 50 μg/ml or 100 μg/ml, more preferably at least 500 μg/ml, andmost preferably at least 1,000 μg/ml in an aqueous solvent. Whether apolypeptide is soluble in an aqueous solution can be readily determinedby a skilled artisan based on its amino acid sequence or through routineexperimentation. Examples of soluble polypeptides of the presentinvention include those that contain all or part of the luminal domainof an SV2 protein but lack at least part of and preferably the entireadjacent transmembrane domain(s). Soluble polypeptides are typicallymore suitable than insoluble polypeptides for intravenousadministration.

The isolated polypeptide of the invention can include one or more aminoacids at either or both N-terminal and C-terminal ends of aBoNT/A-binding sequence of an SV2 protein, where the additional aminoacid(s) do not materially affect the BoNT/A binding function. Anyadditional amino acids can, but need not, have advantageous use inpurifying, detecting, or stabilizing the polypeptide.

In order to improve the stability and/or binding properties of apolypeptide, the molecule can be modified by the incorporation ofnon-natural amino acids and/or non-natural chemical linkages between theamino acids. Such molecules are called peptidomimics (H. U. Saragovi etal., Bio/Technology 10:773-778, 1992; S. Chen et al., Proc. Nat'l. Acad.Sci. USA 89:5872-5876, 1992). The production of such compounds isrestricted to chemical synthesis. It is understood that a polypeptide ofthe present invention can be modified into peptidomimics withoutabolishing its function. This can be readily achieved by a skilledartisan.

In another aspect, the present invention relates to an isolated nucleicacid containing a coding polynucleotide or its complement wherein thecoding polynucleotide has an uninterrupted coding sequence that encodesa polypeptide of the invention as set forth above. A nucleic acidcontaining a polynucleotide that can hybridize to the codingpolynucleotide or its complement, under either stringent or moderatelystringent hybridization conditions, is useful for detecting the codingpolypeptide and thus is within the scope of the present invention.Stringent hybridization conditions are defined as hybridizing at 68° C.in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1%SDS+/−100 μg/ml denatured salmon sperm DNA at room temperature, andmoderately stringent hybridization conditions are defined as washing inthe same buffer at 42° C. Additional guidance regarding such conditionsis readily available in the art, for example, by Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.;and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10. A nucleic acid containing apolynucleotide that is at least 80%, 85%, 90%, or 95% identical to thecoding polynucleotide or its complement over the entire length of thecoding polynucleotide can also be used as a probe for detecting thecoding polynucleotide and is thus within the scope of the presentinvention. Specifically excluded from the present invention is a nucleicacid that contains a nucleotide sequence encoding a full length SV2protein. In one embodiment, a nucleic acid that consists of apolynucleotide that encodes an SV2 luminal domain and a nucleic acidthat comprises a polynucleotide that encodes a polypeptide having an SV2luminal domain wherein the domain is flanked at one or both ends by anon-native amino acid sequence are excluded.

In a related aspect, any nucleic acid of the present invention describedabove can be provided in a vector in a manner known to those skilled inthe art. The vector can be a cloning vector or an expression vector. Inan expression vector, the polypeptide-encoding polynucleotide is underthe transcriptional control of one or more non-native expression controlsequences which can include a promoter not natively found adjacent tothe polynucleotide such that the encoded polypeptide can be producedwhen the vector is provided in a compatible host cell or in a cell-freetranscription and translation system. Such cell-based and cell-freesystems are well known to a skilled artisan. Cells comprising a vectorcontaining a nucleic acid of the invention are themselves within thescope of the present invention. Also within the scope of the presentinvention is a host cell having the nucleic acid of the presentinvention integrated into its genome at a non-native site.

Ligand-Polypeptide Complexes

In another aspect, the present invention relates to a complex of aligand and a polypeptide, wherein the polypeptide comprises a member towhich the ligand binds, the member being selected from (i) amino acids529-562 of rat SV2C, (ii) amino acids 486 to 519 of rat SV2B, (iii)amino acids 543 to 576 of rat SV2A, (iv) a fragment of a homolog of therat SV2C, SV2B, or SV2A wherein the fragment corresponds to amino acids529-562 of rat SV2C, amino acids 486 to 519 of rat SV2B, or amino acids543 to 576 of rat SV2A, respectively, (v) amino acids 454-546 of ratSV2C, (vi) amino acids 411 to 503 of rat SV2B, (vii) amino acids 468 to560 of rat SV2A, (viii) a fragment of a homolog of the rat SV2C, SV2B,or SV2A wherein the fragment corresponds to amino acids 454-546 of ratSV2C, amino acids 411 to 503 of rat SV2B, or amino acids 468 to 560 ofrat SV2A, respectively, (ix) an amino acid sequence that is at least 70%identical to any of the amino acid sequences in (i) to (viii) and iscapable of binding to BoNT/A, and (x) an amino acid sequence from (i) to(viii) with conservative substitutions and is capable of binding toBoNT/A, with the proviso that where the polypeptide is a full length SV2protein, the ligand is not a botulinum toxin. The complexes disclosedherein include both those formed in vitro and in vivo.

In one embodiment, the polypeptide in the complex is a full length SV2protein.

In another embodiment, the polypeptide in the complex is one of theBoNT/A-binding polypeptides of the present invention provided in thesection of “polypeptides, polynucleotides, vectors, and host cells.”

In a preferred embodiment, the polypeptide in the complex comprises amember selected from (i) amino acids 529-566 of rat SV2C, (ii) aminoacids 486 to 523 of rat SV2B, (iii) amino acids 543 to 580 of rat SV2A,(iv) a fragment of a homolog of the rat SV2C, SV2B, or SV2A wherein thefragment corresponds to amino acids 529-566 of rat SV2C, amino acids 486to 523 of rat SV2B, or amino acids 543 to 580 of rat SV2A, respectively,(v) amino acids 454-546 of rat SV2C, (vi) amino acids 411 to 503 of ratSV2B, (vii) amino acids 468 to 560 of rat SV2A, and (viii) a fragment ofa homolog of the rat SV2C, SV2B, or SV2A wherein the fragmentcorresponds to amino acids 454-546 of rat SV2C, amino acids 411 to 503of rat SV2B, or amino acids 468 to 560 of rat SV2A, respectively.

The polypeptide in the complex may be a synthetic or recombinant peptideand it may contain an affinity tag and/or a ganglioside binding site.

In one embodiment, the ligand in the complex is an antibody against thepolypeptide or a BoNT/A fragment that binds to the polypeptide. Such anantibody and BoNT/A fragment can reduces the binding between thepolypeptide and BoNT/A.

Methods for Reducing BoNT/A Neuro-Toxicity

In another aspect, the present invention relates to a method forreducing BoNT/A cellular toxicity in target cells such as neurons. As aresult, botulism disease can be prevented or treated. In one embodiment,the method is used to reduce BoNT/A toxicity in a human or non-humananimal by administering to the human or non-human animal an agent thatcan reduce BoNT/A toxicity.

The term “reducing BoNT/A cellular toxicity” encompasses any level ofreduction in BoNT/A toxicity. The BoNT/A toxicity can be reduced byreducing the level of an SV2 protein in target cells, by inhibitingBoNT/A-related cellular functions of an SV2 protein in target cells, orby reducing the binding between BoNT/A and an SV2 protein located on thecellular surface of target cells. The binding between BoNT/A and an SV2protein can be reduced by either blocking the binding directly or byreducing the amount of SV2 proteins available for binding.

There are many methods by which cellular protein levels such as thelevel of an SV2 protein can be reduced. The present invention is notlimited to a particular method in this regard. As an example, thecellular level of an SV2 protein can be reduced by using the antisensetechnology. For instance, a 20-25 mer antisense oligonucleotide directedagainst the 5′ end of an SV2 mRNA can be generated. Phosphorothioatederivatives can be employed on the last three base pairs on the 3′ and5′ ends of the antisense oligonucleotide to enhance its half-life andstability. A carrier such as a cationic liposome can be employed todeliver the antisense oligonucleotide. In this regard, theoligonucleotide is mixed with the cationic liposome prepared by mixing1-alpha dioleylphatidylcelthanolamine with dimethldioctadecylammoniumbromide in a ratio of 5:2 in 1 ml of chloroform. The solvent isevaporated and the lipids resuspended by sonication in 10 ml of saline.Another way to use an antisense oligonucleotide is to engineer it into avector so that the vector can produce an antisense cRNA that blocks thetranslation of an SV2 mRNA. Similarly, RNAi techniques, which are nowbeing applied to mammalian systems, are also suited for inhibiting theexpression of an SV2 protein. (See Zamore, Nat. Struct. Biol. 8:746-750,2001, incorporated herein by reference as if set forth in its entirety).

Dominant Negative SV2

In another aspect, the present invention relates to identifying adominant negative SV2 that can negate the effects of BoNT/A on cellsthat express the corresponding wild-type SV2. A dominant negative SV2can be identified by introducing a mutation into a wild-type SV2 gene,expressing the mutated SV2 and the wild-type SV2 in the same host celland determining the effect of the mutated SV2 on parameters that relateto BoNT/A toxicity, which include but are not limited to susceptibilityof the host cell to BoNT/A, integration of newly formed SV2 into thehost cell membrane, binding of wild-type SV2 to BoNT/A, and uptake ofBoNT/A into the cells. The wild-type SV2 expressed in the host cell canbe the endogenous SV2 or an SV2 introduced into the host cell. Anydominant negative SV2 identified is within the scope of the presentinvention. The identified dominant negative SV2 can be used to negatethe effect of BoNT/A.

Blocking the Binding Between BoNT/A and SV2

The identification of SV2 as the BoNT/A receptor as well as theBoNT/A-binding sequences on SV2 enable those skilled in the art to blockthe binding between BoNT/A and its receptor through many strategiesavailable in the art. One strategy involves the use of monoclonal andpolyclonal antibodies specific for the BoNT/A-binding sequences on SV2.It is well within the capability of a skilled artisan to generate suchmonoclonal and polyclonal antibodies. The antibodies so generated arewithin the scope of the present invention.

Another strategy involves the use of a BoNT/A-binding polypeptide,preferably a soluble BoNT/A-binding polypeptide, to compete with thereceptor for BoNT/A binding. For example, the BoNT/A-binding polypeptideof the present invention described above in the section of“polypeptides, polynucleotides, vectors, and host cells” can be employedfor this purpose. Other polypeptides that can be employed include thosethat comprise a full length SV2 protein, those that consist of an SV2luminal domain, and those that comprise an SV2 luminal domain whereinthe domain is flanked at one or both ends by a non-native flanking aminoacid sequence.

To block the binding between BoNT/A and its receptor in an animal (humanor non-human), a BoNT/A-binding polypeptide from both the same and adifferent species can be used. The polypeptide can be introduced intothe animal by administering the polypeptide directly or by administeringa vector that can express the polypeptide in the animal.

Those skilled in the art understand that mutations such assubstitutions, insertions and deletions can be introduced into aBoNT/A-binding sequence of an SV2 protein without abolishing theirBoNT/A binding activity. Some mutations may even enhance the bindingactivity. A polypeptide containing such modifications can be used in themethod of the present invention. Such polypeptides can be identified byusing the screening methods described below.

In addition, as gangliosides may promote formation of stable BoNT/A-SV2complexes, the binding between BoNT/A and an SV2 protein may be reducedthrough reducing the binding between the gangliosides and the SV2protein or through reducing the amount of gangliosides available forbinding to the SV2 protein. In a related aspect, when a BoNT/A-bindingpolypeptide is used for reducing BoNT/A toxicity by forming a complexwith BoNT/A, gangliosides may be included to facilitate the formation ofthe complex.

Identifying Agents that can Block Binding Between BoNT/A and SV2

Agents that can block binding between BoNT/A and SV2 can be screened byemploying BoNT/A and a polypeptide that contains a BoNT/A-bindingsequence of an SV2 protein under the conditions suitable for BoNT/A tobind the polypeptide. Gangliosides are optionally included in thereaction mixture. The binding between BoNT/A and the polypeptide can bemeasured in the presence of a test agent and compared to that of acontrol that is not exposed to the test agent. A lower than controlbinding in the test group indicates that the agent can block bindingbetween BoNT/A and the SV2 protein. Other BoNT/A-binding polypeptidesthat can be employed in the method include those of the presentinvention as described above in the section of “polypeptides,polynucleotides, vectors, and host cells.”

There are many systems with which a skilled artisan is familiar forassaying the binding between BoNT/A and a BoNT/A-binding polypeptide.Any of these systems can be used in the screening method. Detailedexperimental conditions can be readily determined by a skilled artisan.For example, the binding between BoNT/A and the polypeptide describedabove can be measured in vitro (cell free system). A cell culture systemin which an SV2 protein is expressed and translocated onto the cellularmembrane can also be used. For the cell culture system, in addition tothe binding between BoNT/A and the SV2 protein, the cellular entry ofBoNT/A and a number of other parameters can also be used as an indicatorof binding between BoNT/A and SV2.

Any method known to one of ordinary skill in the art for measuringprotein-protein interaction can be used to measure the binding betweenBoNT/A and a BoNT/A-binding polypeptide. Coimmunoprecipitation andaffinity column isolation are two commonly used methods.

Surface plasmon resonance (SPR) is another commonly used method. SPRuses changes in refractive index to quantify binding and dissociation ofmacromolecules to ligands covalently linked onto a thin gold chip withina micro flow cell. This technique has been used to study protein-proteininteractions in many systems, including the interactions of PA63 with EFand LF (Elliott, J. L. et al., Biochemistry 39:6706-6713, 2000). Itprovides high sensitivity and accuracy and the ability to observebinding and release in real time. Besides the equilibrium dissociationconstant (Kd), on- and off-rate constants (ka and kd) may also beobtained. Typically, a protein to be studied is covalently tethered to acarboxymethyl dextran matrix bonded to the gold chip. Binding of aproteinaceous ligand to the immobilized protein results in a change inrefractive index of the dextran/protein layer, and this is quantified bySPR. A BIAcore 2000 instrument (Pharmacia Biotech) can be used for thesemeasurements.

For the cell culture system, the binding of BoNT/A to a BoNT/A-bindingpolypeptide can be assayed by staining the cells, the examples of whichare described in the example section below.

Identifying Agents that can Bind to a BoNT/A-Binding Sequence of SV2

Agents that can bind to a BoNT/A-binding sequence of an SV2 protein canbe used to block the binding between BoNT/A and the SV2 protein. Suchagents can be identified by providing a polypeptide that contains aBoNT/A-binding sequence of an SV2 protein to a test agent, anddetermining whether the agent binds to the BoNT/A-binding sequence.Other BoNT/A-binding polypeptides that can be employed in the methodinclude those of the present invention as described above in the sectionof “polypeptides, polynucleotides, vectors, and host cells.” Any agentidentified by the method can be further tested for the ability to blockBoNT/A entry into cells or to neutralize BoNT/A toxicity. A skilledartisan is familiar with the suitable systems that can be used for thefurther testing. Examples of such systems are provided in the examplesection below.

The skilled artisan is familiar with many systems in the art forassaying the binding between a polypeptide and an agent. Any of thesesystems can be used in the method of the present invention. Detailedexperimental conditions can be readily determined by a skilled artisan.For example, a polypeptide that contains a BoNT/A-binding sequence of anSV2 protein can be provided on a suitable substrate and exposed to atest agent. The binding of the agent to the polypeptide can be detectedeither by the loss of ability of the polypeptide to bind to an antibodyor by the labeling of the polypeptide if the agent is radioactively,fluorescently, or otherwise labeled. In another example, a polypeptidethat contains a BoNT/A-binding sequence of an SV2 protein can beexpressed in a host cell, and the cell is then exposed to a test agent.Next, the polypeptide can be isolated, e.g., by immunoprecipitation orelectrophoresis, and the binding between the polypeptide and the agentcan be determined. As mentioned above, one way to determine the bindingbetween the polypeptide and the agent is to label the agent so that thepolypeptide that binds to the agent becomes labeled upon binding. If thetest agent is a polypeptide, examples of specific techniques forassaying protein/protein binding as described above can also be used. Itshould be noted that when a BoNT/A-binding sequence of an SV2 proteinused in the screening assay have flanking sequences, it may be necessaryto confirm that an agent binds to the BoNT/A-binding sequence ratherthan the flanking sequences, which can be readily accomplished by askilled artisan.

Agents that can be Screened

The agents screened in the above screening methods can be, for example,a high molecular weight molecule such as a polypeptide (including, e.g.,a polypeptide containing a modified BoNT/A-binding sequence of an SV2protein, or a monoclonal or polyclonal antibody against a BoNT/A-bindingsequence of an SV2 protein), a polysaccharide, a lipid, a nucleic acid,a low molecular weight organic or inorganic molecule, or the like.

Batteries of agents for screening are commercially available in the formof various chemical libraries including peptide libraries. Examples ofsuch libraries include those from ASINEX (i.e. the Combined WisdomLibrary of 24,000 manually synthesized organic molecules) and CHEMBRIDGECORPORATION (i.e. the DIVERSet™ library of 50,000 manually synthesizedchemical compounds; the SCREEN-Set™ library of 24,000 manuallysynthesized chemical compounds; the CNS-Set™ library of 11,000compounds; the Cherry-Pick™ library of up to 300,000 compounds) andlinear library, multimeric library and cyclic library (Tecnogen(Italy)). Once an agent with desired activity is identified, a libraryof derivatives of that agent can be screened for better molecules. Phagedisplay is also a suitable approach for finding novel inhibitors of theinteraction between BoNT/A and SV2.

Methods of Detecting BoNT/A or Clostridium botulinum

In another aspect, the present invention relates to a method ofdetecting BoNT/A or Clostridium botulinum. The method involves exposinga sample suspected of containing BoNT/A to an agent that contains apolypeptide having a BoNT/A-binding sequence of an SV2 protein, anddetecting binding of the polypeptide to BoNT/A. Other BoNT/A-bindingpolypeptides that can be employed in the method include those of thepresent invention as described above in the section of “polypeptides,polynucleotides, vectors, and host cells.”

Methods for Identifying Polypeptides that can Bind to BoNT/A

In another aspect, the present invention relates to a method foridentifying polypeptides that can bind to BoNT/A. The method involvesproviding a polypeptide that comprises a BoNT/A-binding sequence of anSV2 protein, modifying the polypeptide at the BoNT/A-binding sequence,and determining whether the modified polypeptide can bind to BoNT/A.

Kits

Any product of the invention described herein can be combined with oneor more other reagent, buffer or the like in the form of a kit (e.g., adiagnosis, prevention, or treatment kit) in accord with theunderstanding of a skilled artisan.

The invention will be more fully understood upon consideration of thefollowing non-limiting example.

Example

In this example, we demonstrate that BoNT/A binds to all three SV2isoforms (SV2A, SV2B, and SV2C). Particular binding fragments such asamino acids 529-562, 529-566, and 454-546 of the rat SV2C were alsoidentified. Recombinant SV2 fragments inhibit BoNT/A binding tohippocampal neurons and motor nerve terminals. Significantly, BoNT/Abinding to hippocampal neurons was abolished in SV2A/B knockout mice andthis binding can be restored by transfecting neurons with SV2.Consistently, BoNT/A binding was reduced at diaphragm motor nerveterminals in SV2 knockout mice, and SV2B knockout mice displayed reducedsensitivity to BoNT/A. These data establish SV2 as the protein receptorfor BoNT/A, which mediates toxin entry through synaptic vesiclerecycling.

Materials and Methods

Materials, antibodies and SV2 knockout mouse lines: Alexa 488-conjugatedα-BTX was purchased from Molecular Probes, Inc. (OR). A mAb thatrecognizes SNAP-25 after it has been cleaved by BoNT/A (anti-SNAP-25-C)was purchased from Research & Diagnostic Antibodies, Inc. (CA). mAbsdirected against SV2 (pan-SV2), Syp (Cl7.2), Syb II (Cl69.1) and Syt I(Syt I_(N) Ab, Cl604.4) were generously provided by R. Jahn(Max-Planck-Institute for Biophysical Chemistry, Gottingen, Germany). Ahuman antibody directed against BoNT/A (RAZ-1) was generously providedby J. Marks (University of California—San Francisco, Calif.). Cy2, Cy3,Cy5, Alexa 546 and Alexa 647 conjugated secondary antibodies werepurchased from Jackson Laboratories (ME) and Molecular Probes, Inc.Rabbit polyclonal anti-BoNT/A, B and E antibodies and anti-SV2A, B and Cantibodies were described in Dong M et al., J. Cell. Biol.162:1293-1303, 2003; and Janz R and Sudhof T C, Neuroscience94:1279-1290, 1999, both are herein incorporated by reference in theirentirety). BoNT/A, B and E were purified as described in Malizio C G,Methods and Protocols, O. Hoist, ed. (Humana Press), pp. 27-39, 2000,which is incorporated by reference in its entirety. A mixture of bovinebrain gangliosides was purchased from Matreya LLC (PA). The SV2 knockoutmouse lines used in this study were described in Janz R et al., Neuron24:1003-1016, 1999, which is incorporated by reference in its entirety.Mice were genotyped by PCR as described in Janz R et al., supra, 1999.

cDNA, constructs and transfection: Rat SV2A, B and C cDNAs weredescribed in Bajjalieh S M et al. Science 257: 1271-3, 1992; Feany M Bet al. Cell 70: 861-7, 1992; Bajjalieh S M et al. Proc Natl Acad Sci USA90: 2150-4, 1993; and Janz R & Sudhof T C Neuroscience 94: 1279-90,1999, all of which are herein incorporated by reference in theirentirety. Various SV2 luminal domain fragments were generated by PCR,subcloned into pGEX-2T and purified as GST fusion proteins (Lewis J L etal. J Biol Chem 276: 15458-65, 2001). GST and GST tagged SV2C-L4proteins were also purified using magnetic GST beads according to themanufacturers protocol (Promega, Wis.), eluted with 40 mM Glutathione(Sigma), and subsequently dialyzed to produce high concentrations ofsoluble protein.

To transfect hippocampal neurons with SV2 isoforms, full length SV2A, Band C were subcloned into the Lox-Syn-Syn lentivirus vector (provided byP. Scheiffele, Columbia University, NY). This vector is a modifiedversion of pFUGW (Lois C et al., Science 295:868-872, 2002) and containsseparate neuronal-specific (synapsin) promoters. One promoter controlsthe expression of SV2 isoforms inserted between BamHI and NotI sites andthe other promoter controls expression of EGFP to detect transfectedcells. Transfections were performed on neurons 7-10 DIV usingLipofectamine 2000 (Invitrogen) as described in Dean C et al., Nat.Neurosci. 6:708-716, 2003 (incorporated by reference in its entirety)and analyzed 48 hrs later. Note: The BamHI site inside the SV2C sequencehas been mutated (GGATCC to GGATAC, preserving the amino acid sequence)to simplify subcloning.

Neuronal cell cultures, BoNT uptake, immunocytochemistry: Cultures ofhippocampal neurons were prepared from E18-19 rats, and SV2 knockoutmouse neuron cultures were prepared from P1 mice. Neurons were plated onpoly-D-lysine coated glass coverslips (12 mm) at a density of 50,000/cm²and cultured in Neurobasal medium supplemented with B-27 (2%) andGlutamax (2 mM). Experiments were carried out on neurons 10-14 days old.

To assay for BoNT/A uptake under different conditions (FIG. 1 b),hippocampal neurons were incubated in one of the following assay buffers(200 μl) containing BoNT/A (10 nM) and Syt I_(N) Ab (604.4, 1:40) for 1min. These buffers are: control buffer (PBS: 140 mM NaCl, 3 mM KCl, 1.5mM KH₂PO₄, 8 mM Na₂HPO₄, 1 mM CaCl₂, 0.5 mM MgCl₂), high K⁺ (same ascontrol buffer but adjusted to 56 mM KCl and 87 mM NaCl), and high K⁺/NoCa²⁺ buffer (same as high K⁺ buffer but lacking CaCl₂). Neurons werethen washed in PBS (3×500 μl) and fixed in 4% paraformaldehyde for 15min. After permeabilization with 0.3% Triton X-100, neurons were blockedwith 10% goat serum and stained with a polyclonal anti-BoNT/A antibody(1:200) for 1 hr at room temperature. The secondary antibodies wereCy2-conjugated goat-anti-mouse and Cy3-conjugated goat-anti-rabbitImmunofluorescence images were acquired using a confocal microscope(Olympus FV1000, 60× water-immersion objective). Identical gain andlaser settings were used for images that were directly compared in thefigures. For all experiments using hippocampal neurons after FIG. 1 b,neurons were incubated in high K⁺ buffer for 10 min in order to increasethe amount of toxin entry.

For triple staining of BoNT/A, BoNT/B and SV2 (FIG. 4 b), BoNT/B wasdetected with a rabbit polyclonal antibody (1:200) and a Cy2-conjugatedsecondary antibody; BoNT/A was detected with a human antibody (RAZ-1,1:300) and Alexa-546 conjugated secondary antibody; and SV2 expressionwas detected with a mouse monoclonal antibody (pan-SV2 Ab, 1:400), andAlexa-647 conjugated secondary antibody.

To detect SNAP-25 that has been cleaved by BoNT/A (FIG. 3 b), neuronswere washed three times after exposure to BoNT/A, and further incubatedin culture media for 6 hrs. Cells were then fixed, permeabilized, andstained with the anti-SNAP-25-C monoclonal antibody (1:50) and therabbit anti-BoNT/A antibody.

ImageJ software (NIH) was used to quantify fluorescence intensitiesshown in FIG. 4. Briefly, a fixed threshold was first chosen for eachchannel (BoNT/B and BoNT/A) to exclude background signals from regionslacking neurons, and the average intensity of the fluorescence signalsfrom decorated neurons was measured. Neurons that were not exposed toBoNT/A and B were fixed and stained with the same antibodies inparallel. The average intensity of these images was subtracted fromsamples treated with the toxins. Two-tailed t tests were used todetermine statistical significance.

Co-immunoprecipitation and pull-down assays: Rat or mice brain detergentextracts were made as described in Lewis J L et al., J. Biol. Chem.276:15458-15465, 2001, which is incorporated by reference in itsentirety. BoNT/A was premixed with brain extracts (400 μl, 3-6 mg/ml)for 1 hr at 4° C. before adding antibodies (5 μl), and then furtherincubated for 1 hr. Protein G Fast Flow beads (40 μl, AmershamBiosciences) were added and incubated for 1 hr. Beads were washed threetimes in TBS (20 mM Tris, 150 mM NaCl, pH 7.4) plus 0.5% Triton X-100.Bound material was subjected to SDS-PAGE and western blot analysis.

Recombinant GST fusion proteins were purified and immobilized onglutathione-Sepharose beads. Pull down assays were carried out asdescribed in Dong M et al. (supra, 2003), using 8 μg immobilizedproteins and 100 nM toxins in 100 μl TBS plus 0.5% Triton X-100. Boundmaterial was subjected to SDS-PAGE and western blot analysis.

Mouse hemi-diaphragm experiments: Mouse hemi-diaphragms were kept ineither control buffer (FIG. 1 a, mammalian Ringer, in mM: NaCl 138.8,KCl 4, NaHCO₃ 12, KH₂PO₄ 1, MgCl₂ 2, CaCl₂ 2, and glucose 11), or highK⁺ buffer (same as control buffer but adjusted to 98 mM NaCl and 45 mMKCl), warmed to 37° C. and gased with 95% O₂/5% CO₂. Hemi-diaphragmswere incubated with the indicated BoNTs (25 nM) for 1 hr at roomtemperature (note: 10 nM BoNT/A was used and incubated for only 30 minin FIG. 3 c). After incubation, diaphragms were washed and fixed with 4%paraformaldehyde for 30 min at room temperature, permeabilized andblocked in 5% goat serum plus 0.5% Triton X-100. Diaphragms wereincubated with Alexa-488-conjugated α-BTX (1:250) and rabbit anti-BoNT/Aor B antibodies (1:1000) at 4° C., overnight. A Cy3-conjugatedanti-rabbit secondary antibody was used at a dilution of 1:800. Forstaining SV2A, B or C in the NMJ (FIG. 8), the hemi-diaphragms wereexcised and immediately fixed. A 1:1000 dilution of the specific rabbitanti-SV2A, B or C antibodies were used. All images were collected usinga confocal microscope (Olympus FV1000, 60× water-immersion objective).

To quantify fluorescent signals, the α-BTX channel was pseudo-coloredgreen and BoNT/A (or BoNT/B) channel was pseudo-colored red. Mergedgreen and red images were imported into MetaMorph software(Improvision). The regions of interests (ROI) marking NMJs weredetermined by thresholding the α-BTX green channel. The same thresholdvalues were used throughout the diaphragm experiments. The averageintensity of green and red channels within ROIs were measured and theratio between them used to determine the level of toxin binding.Two-tailed t tests were used to determine statistical significancebetween pairwise sets of data.

Rapid BoNT toxicity assay in mice: BoNT/A effective toxicity in mice wasestimated using the intravenous method described in Boroff D A and FleckU, J. Bacteriol. 92:1580-1581, 1966 (incorporated by reference in itsentirety); Dong M et al., supra, 2003; and Malizio C G, supra, 2000.Briefly, BoNT/A (type A1) isolated from Hall strain was diluted to 10μg/ml in 30 mM sodium phosphate buffer (pH 6.3 plus 0.2% gelatin). Eachmouse was injected intravenously (lateral tail vein) with 0.1 ml of thediluted toxin and their time-to-death was recorded. The time-to-deathvalues were converted to intraperitoneal LD₅₀/ml using a standard curvedescribed in Malizio C G, supra, 2000. SV2B knockout mice used in theseexperiments had been crossed for 6 generation against C57B16/J mice.

Results

The BoNT/A receptor resides on synaptic vesicles: The physiologicaltarget for BoNT/A is peripheral motor nerve terminals (Dolly J O et al.,Nature 307:457-460, 1984). Stimulation of neuronal activity (i.e.neurotransmitter release) can accelerate the rate of paralysis caused byBoNT/A (Hughes R W, J. Physiol. (Lond.) 160:221-233, 1962). However, itis not known whether synaptic vesicle exocytosis directly increasesBoNT/A binding and entry into neurons. To address this question, wevisualized BoNT/A binding to motor nerve terminals in mouse diaphragmpreparations. The neuromuscular junctions (NMJs) in this tissue werelabeled with α-bungarotoxin (α-BTX), which binds to postsynapticacetylcholine receptors (Astrow S H et al., J. Neurosci. 12:1602-1615,1992). As shown in FIG. 1 a, high K⁺ solution (45 mM KCl), whichtriggers synaptic vesicle exocytosis, increased BoNT/A binding to NMJsby approximately 6-fold compared to control conditions, indicating thatsynaptic vesicle exocytosis exposes BoNT/A receptors.

To further analyze whether the BoNT/A receptor is on synaptic vesicles,we used cultured rat hippocampal neurons as a model system. Synapticvesicle recycling was monitored through the uptake of an antibody thatrecognizes the luminal domain of synaptotagmin I (Syt I_(N) Ab) (Dong Met al., J. Cell. Biol. 162:1293-1303, 2003), an abundant synapticvesicle membrane protein. As shown in FIG. 1 b, uptake of Syt I_(N) Abwas greatly increased by a short stimulation with high K⁺ (56 mM KCl, 1min), and inhibited by depletion of extracellular Ca²⁺. Interestingly,uptake of BoNT/A to the same neurons mimicked Syt I_(N) Ab behavior andlargely co-localize with Syt I_(N) Ab signals (FIG. 1 b). To furtherconfirm this finding, we pretreated these neurons with BoNT/B, whichspecifically blocks synaptic vesicle exocytosis by cleaving Syb II, asynaptic vesicle membrane protein required for exocytosis (Schiavo G etal., Nature 359:832-835, 1992). As shown in FIG. 1 c, BoNT/B treatmentabolished uptake of BoNT/A under stimulated conditions, indicating thatthe BoNT/A receptor resides on vesicles containing Syb II in neurons.Together, these evidences suggest that the receptor for BoNT/A is onsynaptic vesicles.

BoNT/A binds to the luminal domain of SV2: Synaptic vesicles arewell-studied organelles, and most, if not all, integral synaptic vesicleproteins have been identified (Fernandez-Chacon R and Sudhof T C Ann.Rev. Physiol. 61:753-776, 1999). We screened known synaptic vesiclemembrane proteins for BoNT/A interactions by using their specificantibodies to co-immunoprecipitate BoNT/A from rat brain detergentextracts. As shown in FIG. 2 a, an SV2 monoclonal antibody (pan-SV2) wasable to immunoprecipitate BoNT/A. This interaction is specific since anantibody against synaptophysin (Syp), another abundant vesicle protein,failed to pull down significant amounts of BoNT/A (FIG. 2 a).

We next assessed whether BoNT/A-SV2 interactions are affected bygangliosides. We increased ganglioside concentration in brain detergentextracts by adding exogenous gangliosides. Immunoprecipitations wereperformed at various BoNT/A concentrations. As indicated in FIG. 2 b,adding exogenous gangliosides increased the level ofco-immunoprecipitation of BoNT/A. This effect was not significant athigh BoNT/A concentration (100 nM), but became more apparent at lowtoxin concentration (e.g., 20 nM), indicating gangliosides may promoteformation of stable BoNT/A-SV2 complexes at low toxin concentration.

SV2 is a conserved membrane protein on synaptic vesicles and endocrinesecretory vesicles in vertebrates (Buckley K and Kelly R B, J. Cell.Biol. 100:1284-1294, 1985; Lowe A W et al., J. Cell. Biol. 106:51-59,1988). Three highly homologous isoforms have been identified, denoted asSV2A, B and C (Bajjalieh S M et al., Proc. Natl. Acad. Sci. USA90:2150-2154, 1993; Bajjalieh S M et al., Science 257:1271-1273, 1992;Feany M B et al., Cell 70:861-867, 1992; Janz R and Sudhof T C,Neuroscience 94:1279-1290, 1999). SV2A and B are widely expressedthroughout the brain, while the expression of SV2C is more restricted toevolutionarily older brain regions (Bajjalieh S M et al., J. Neurosci.14:5223-5235, 1994; Janz R and Sudhof T C, supra, 1999). The antibodyused in FIG. 2 a recognizes all three isoforms (Lowe A W et al., supra,1988). As depicted in FIG. 2 d, SV2 isoforms share a similar topology,containing 12 putative transmembrane domains and only one relativelylarge luminal domain (luminal domain 4, L4) (Janz R and Sudhof T C,supra, 1999). Because the luminal domain of SV2 is the only regionexposed to the outside of cells after vesicle exocytosis, we firstexamined whether BoNT/A binding is mediated by SV2 luminal domains. Themajor luminal domain (L4) of SV2A, B and C were purified as GST fusionproteins, immobilized on beads and tested for their ability to pull downBoNT/A, B and E from solution. As shown in FIG. 2 c, we observed directbinding of BoNT/A, but not BoNT/B or E, to all SV2 isoforms. SV2C showedthe most robust binding, and SV2B pulled down the least amount ofBoNT/A.

To determine the critical BoNT/A binding region, we made a series oftruncation mutants within the SV2C-L4 region. As shown in FIG. 2 e, ashort fragment (amino acids 529-566) was able to pull down similarlevels of BoNT/A to the full L4 region. Sequence alignment showed thatthis region, indicated in FIG. 2 d by arrows, is relatively conservedamong SV2 isoforms and includes two putative N-glycosylation sites. Itwas further observed that a shorter fragment, amino acids 529-562, wasalso able to pull down BoNT/A, although not as effectively as the529-566 fragment. In addition, various fragments containing amino acids454-546 were also able to pull down BoNT/A.

SV2C luminal fragments inhibit BoNT/A binding to neurons: Among thethree SV2 isoforms, hippocampal neurons were found to express SV2A andB, but not SV2C (Bajjalieh S M et al., supra, 1994; Janz R and Sudhof TC, supra, 1999). To determine whether SV2 mediates BoNT/A binding inthese neurons, we used soluble recombinant SV2C-L4 fragments, whichshowed the highest apparent affinity for BoNT/A, to inhibit BoNT/Abinding to neurons by competing with endogenous SV2A/B. Neurons wereexposed to BoNT/A and Syt I_(N) Ab for 10 min in the presence of anexcess amount of either control protein (GST) or GST-tagged SV2C-L4fragment. As shown in FIG. 3 a, SV2C-L4 reduced BoNT/A binding toneurons compared to GST. Neurons in both conditions showed similar levelof Syt I_(N) Ab uptake, indicating that the reduction in BoNT/A bindingis not due to nonspecific changes in synaptic vesicle recycling.

To further demonstrate the specificity of this inhibition, we tested inparallel another BoNT, BoNT/B, which has been shown to use the synapticvesicle protein synaptotagmin I/II to enter cells (Dong M et al., supra,2003; Nishiki T et al., J. Biol. Chem. 269:10498-10503, 1994; Nishiki Tet al., Biochim. Biophys. Acta 1158:333-338, 1993). BoNT/B is an idealcontrol since it has similar structure and size to BoNT/A and uses thesame entry pathway. As shown in FIG. 3 a (right panel), SV2C-L4 did notaffect BoNT/B binding to neurons. Furthermore, binding of BoNT/B can beinhibited by adding a peptide (P21) derived from its receptor,synaptotagmin II (Syt II) (Dong M et al., supra, 2003), and addition ofthis peptide did not affect BoNT/A binding (FIGS. 7 a and b).Interestingly, SV2C-L4 also did not affect the binding of BoNT/E,another major BoNT, suggesting BoNT/E does not use SV2 to enter neurons(FIG. 7 c).

We further assessed whether the reduction in BoNT/A binding by additionof SV2C-L4 correlates with the protection of endogenous SNAP-25. Takingadvantage of a specialized antibody, anti-SNAP-25-C, which onlyrecognizes SNAP-25 fragments cleaved by BoNT/A (FIG. 3 b), we monitoredthe level of cleaved fragments by immunostaining Neurons were firsttreated with BoNT/A for 10 min in the presence of either GST or SV2C-L4.These neurons were washed and further incubated for 6 hrs, fixed andimmunostained for cleaved SNAP-25 fragments. As shown in FIG. 3 b,reduced BoNT/A binding by addition of SV2C-L4 resulted in decreasedlevels of SNAP-25 cleavage, indicating that SV2C-L4 prevented thefunctional entry of BoNT/A into neurons.

We extended this study to peripheral motor nerve terminals, thephysiological target of BoNT/A in vivo. Using SV2 isoform specificantibodies, we found that motor nerve terminals at NMJs in the diaphragmexpress all three SV2 isoforms (FIG. 8). Pre-incubation of BoNT/A with ahigh concentration of SV2C-L4 fragments (30 μM) significantly reducedBoNT/A binding to NMJs (65% reduction compared to control, P<0.0001,t-test) (FIGS. 3 c and d). This decrease is specific to BoNT/A sinceSV2C-L4 did not affect BoNT/B binding (FIGS. 3 c and d). These datasuggest that binding of BoNT/A to motor nerve terminals is mediated bydirect interactions with SV2 luminal domains.

BoNT/A binding is abolished in SV2A/B knockout neurons: To determinedefinitively whether SV2 is the receptor for BoNT/A, we turned toavailable SV2A and B single knockout and SV2A/B double knockout mice(Janz R et al., Ann. NY Acad. Sci. 733:345-255, 1999). Mice lacking SV2A(SV2A single knockout and SV2A/B double knockout) display severeseizures and die within 2-3 weeks of birth, while SV2B single knockoutmice are normal. These phenotypes may be because SV2A has widerdistribution than SV2B and these two isoforms are functionally redundant(Janz R et al., supra, 1999). Cultured hippocampal neurons from SV2A/Bknockout mice develop normal synaptic structures and are capable ofreleasing neurotransmitter (Janz R et al., supra, 1999). Because theseneurons only express SV2A and B, neurons from SV2A/B double knockoutmice become an ideal loss-of-function model to study the role of SV2 forBoNT/A binding.

We first tested the function of SV2B by comparing BoNT/A binding toneurons from SV2B knockout (SV2B(−/−)) mice and wild-type littermatecontrols (WT). Neurons were exposed to BoNT/A and B simultaneously for10 min in High K⁺ buffer, washed and fixed. Binding of BoNT/A and B toneurons was quantified by measuring immunofluorescence intensity (seeMethods for details). Normalized average intensities (% WT) are shown inFIG. 4 a. SV2B knockout neurons showed significantly reduced BoNT/Abinding (28% reduction compared to WT, P<0.0001, t-test), while BoNT/Bbinding remained the same. These data suggest that lack of SV2B does notaffect the toxin entry pathway—synaptic vesicle recycling—in general,but rather specifically reduced BoNT/A binding surface binding sites.

Because SV2B(−/−) neurons still express SV2A, we asked whether remainingbinding of BoNT/A is mediated by SV2A. By breeding SV2A(+/−)SV2B(−/−)mice, we generated littermates that have no SV2B but wild-type levels ofSV2A (SV2A(+/+)SV2B(−/−)), no SV2B and half the levels of SV2A(SV2A(+/−)SV2B(−/−)), and SV2A/B double knockouts (SV2A(−/−)SV2B(−/−))(FIG. 4 b). Neurons cultured from these littermates were exposed toBoNT/A and B, washed and fixed. Triple immunostaining of BoNT/A, BoNT/Band SV2 were performed and representative images from each genotype areshown in FIG. 4 b. Quantification of immunofluorescence intensity showedthat BoNT/A binding to SV2A(+/−) SV2B(−/−) neurons is only 47% ofSV2A(+/+)SV2B(−/−) neurons (FIG. 4 c). Interestingly, the majority ofBoNT/A binding in SV2A(+/−)SV2B(−/−) neurons colocalized with SV2Aexpression (FIG. 4 b middle frames). Strikingly, there is virtually nobinding of BoNT/A to SV2A/B double knockout neurons (FIGS. 4 b and c).These changes in binding are specific to BoNT/A, since BoNT/B binding toeach genotype remained the same (FIGS. 4 b and c). This indicates thatSVA/B knockouts specifically abolished BoNT/A recognition sites insteadof causing other entry pathway defects. Together with the fact that SV2Cluminal fragments were able to inhibit BoNT/A binding, we haveestablished that BoNT/A binding to hippocampal neurons is mediated bydirect interactions with SV2A and B.

Expression of SV2 restores BoNT/A binding to SV2A/B knockout neurons:Using hippocampal neurons from SV2A/B double knockout mice, we carriedout rescue studies to determine whether expression of SV2A, B or C canrestore BoNT/A binding. Rat SV2A, B or C were transfected into theseneurons, with a lentiviral vector that can express SV2 and GFPsimultaneously under separated neuronal specific promoters (synapsinpromoter, details described in Methods). Forty eight hrspost-transfection, neurons were exposed to BoNT/A for 10 min, washed,and binding of BoNT/A assessed by immunostaining Transfected neuronswere identified by GFP fluorescence signals and expression of SV2 inthese neurons were confirmed by immunostaining. As shown in FIG. 5,BoNT/A binding was only observed on neurons transfected with SV2A, B orC, while other neurons in the same field showed no binding. Enlargedoverlay images between SV2 and BoNT/A signals showed a high degree ofcolocalization at synapses (FIG. 5, overlay). The high level of SV2expression in the cell soma is likely due to over-expression ofexogenous proteins since it is not found in immunostaining of endogenousSV2 in wild-type neurons. Overexpression of another synaptic vesicleprotein, synaptotagmin I, using the same viral vector did not resultedin detectable BoNT/A fluorescence signals, confirming the specificity ofthe rescue effect. SV2A, B or C all rescued BoNT/A binding, indicatingall three isoforms can mediate BoNT/A binding to neurons once expressed.

BoNT/A binding to motor terminals is reduced in SV2A/B knockout mice: Todetermine whether SV2 function as a receptor for BoNT/A at itsphysiological target, we examined BoNT/A binding to diaphragm nerveterminals from SV2 knockout mice. These nerves normally express allthree SV2 isoforms (FIG. 8) and we expect they all contribute to BoNT/Arecognition of motor terminals. Because SV2A knockout and SV2A/B doubleknockout mice do not survive to adulthood and SV2C knockout mice are notavailable, we compared diaphragm preparations from available adultknockout mice that have the least amount of SV2 expression (SV2A(+/−)SV2B(−/−)), to wild-type (WT). As shown in FIGS. 6 a and b, BoNT/Abinding to NMJs from SV2A(+/−)SV2B(−/−) mice is significantly reducedcompared to WT (72% reduction, P<0.001, t-test), while the levels ofBoNT/B binding are the same (P>0.05, t-test), suggesting that SV2A and Bare important for BoNT/A binding to motor nerve terminals. The remaininglevel of BoNT/A binding in SV2A(+/−)SV2B(−/−) NMJs is likely mediated bySV2C, which is not altered in these mice, and remaining reduced level ofSV2A.

SV2B knockout mice have reduced sensitivity to BoNT/A: To furtherestablish the physiological meaning of these findings, we extended ourstudied to the whole animal. Among available SV2 knockout mice lines,SV2A knockout and SV2A/B double knockout mice both die within weeksafter birth, suggesting SV2A is essential for maintaining normalsynaptic transmission. In contrast, SV2B single knockout mice(SV2B(−/−)) have no apparent difference to wild-type (WT) mice. Tominimize the potential defect in vivo on synaptic transmission, we choseto compare BoNT/A sensitivity of SV2B knockout mice and WT littermates.Sensitivity to BoNT/A was assessed with an established rapid assay, inwhich large amount of toxins are injected intravenously and the survivaltime (Time-to-death) after the injection were recorded (Boroff D A andFleck U, J. Bacteria 92:1580-1581, 1966; Dong M et al., supra, 2003;Malizio C G, Methods and Protocols O. Hoist, ed. (Humana Press), pp.27-39, 2000). This survival time can be converted to apparent toxicityfrom a previously established standard curve (Malizio C G, supra, 2000).

Identical amounts of BoNT/A (10⁴-10⁶ LD₅₀/ml) were injected intoSV2B(−/−) and WT mice and their survival time is shown in FIG. 6 c. SV2Bknockout mice survived significantly longer than wild-type littermates(43.7±1.9 min versus 32.6±4.6 min). The effective toxin concentrationestimated from the survival time of WT mice is about 2.5-fold more thanSV2B(−/−) mice (8.4±2.9×10⁵ LD₅₀/ml versus 3.4±0.5×10⁵ LD₅₀/ml, FIG. 6c). The difference in effective toxicity reflects the shift of LD₅₀value in SV2B knockout mice, i.e., these mice require approximately 2.5fold more BoNT/A for a lethal dose than their WT littermates. Theremaining toxicity in SV2B(−/−) mice is likely mediated by SV2A and C intheir motor nerve terminals. These results provided functional evidencethat SV2 is the physiological receptor for BoNT/A in vivo.

The present invention is not intended to be limited to the foregoingexample, but rather to encompass all such variations and modificationsas come within the scope of the appended claims.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom (i) amino acids 529-566 of rat SV2C (SEQ ID NO:6), (ii) amino acids529-566 of human SV2C (SEQ ID NO:18), (iii) amino acids 486 to 523 ofrat SV2B (SEQ ID NO:4), (iv) amino acids 486 to 523 of mouse SV2B (SEQID NO:10), (v) amino acids 486 to 523 of human SV2B (SEQ ID NO:16), (vi)amino acids 543 to 576 of rat SV2A (SEQ ID NO:2), (vii) amino acids 454to 580 of rat SV2C (SEQ ID NO:6), (viii) amino acids 454 to 580 of humanSV2C (SEQ ID NO:18), (ix) amino acids 411 to 536 of rat SV2B (SEQ IDNO:4), (x) amino acids 411 to 536 of mouse SV2B (SEQ ID NO:10), (xi)amino acids 411 to 536 of human SV2B (SEQ ID NO:16), (xii) amino acids468 to 595 of rat SV2A (SEQ ID NO:2), (xiii) amino acids 468 to 595 ofhuman SV2A (SEQ ID NO:14), and an amino acid sequence that is at least95% identical to (iii), (iv), (v), (ix), (x), or (xi) and is capable ofbinding to botulinum neurotoxin A (BoNT/A).
 2. An antibody that bindsspecifically to the polypeptide of claim
 1. 3. An isolated nucleic acidcomprising a polynucleotide or its complement wherein the polynucleotideis selected from: (1) a first polynucleotide that encodes thepolypeptide of claim 1; and (2) a second polynucleotide that hybridizesto the first polynucleotide under stringent or moderately stringenthybridization conditions, with the proviso that a nucleic acidcomprising a polynucleotide that encodes a full length SV2, a nucleicacid consisting of a polynucleotide that encodes an SV2 luminal domain,and a nucleic acid comprising a polynucleotide that encodes apolypeptide having an SV2 luminal domain wherein the domain is flankedat one or both ends by a non-native amino acid sequence are excluded. 4.The isolated nucleic acid of claim 3, wherein the nucleic acid comprisesa polynucleotide that encodes a polypeptide selected from (i) aminoacids 529-566 of rat SV2C (SEQ ID NO:6), (ii) amino acids 529-566 ofhuman SV2C (SEQ ID NO:18), (iii) amino acids 486 to 523 of rat SV2B (SEQID NO:4), (iv) amino acids 486 to 523 of mouse SV2B (SEQ ID NO:10), (v)amino acids 486 to 523 of human SV2B (SEQ ID NO:16), (vi) amino acids543 to 580 of rat SV2A (SEQ ID NO:2), and (vii) an amino acid sequencethat is at least 95% identical to (iii), (iv), or (v) and is capable ofbinding to botulinum neurotoxin A (BoNT/A).
 5. A vector comprising thenucleic acid of claim 3 operably linked to a non-native promoter.
 6. Ahost cell comprising the nucleic acid of claim 3 operably linked to anon-native promoter.
 7. A complex of a ligand and a polypeptide, whereinthe polypeptide comprises a member to which the ligand binds, the memberbeing selected from (i) amino acids 529-562 of rat SV2C (SEQ ID NO:6),(ii) amino acids 486 to 519 of rat SV2B (SEQ ID NO:4), (iii) amino acids543 to 576 of rat SV2A (SEQ ID NO:2), (iv) a fragment of a homolog ofthe rat SV2C, SV2B, or SV2A wherein the fragment corresponds to aminoacids 529-562 of rat SV2C, amino acids 486 to 519 of rat SV2B, or aminoacids 543 to 576 of rat SV2A, respectively, (v) amino acids 454-546 ofrat SV2C (SEQ ID NO:6), (vi) amino acids 411 to 503 of rat SV2B (SEQ IDNO:4), (vii) amino acids 468 to 560 of rat SV2A (SEQ ID NO:2), (viii) afragment of a homolog of the rat SV2C, SV2B, or SV2A wherein thefragment corresponds to amino acids 454-546 of rat SV2C, amino acids 411to 503 of rat SV2B, or amino acids 468 to 560 of rat SV2A, respectively,(ix) an amino acid sequence that is at least 70% identical to any of theamino acid sequences in (i) to (viii) and is capable of binding toBoNT/A, and (x) an amino acid sequence from (i) to (viii) withconservative substitutions and is capable of binding to BoNT/A, with theproviso that where the polypeptide is a full length SV2 protein, theligand is not a botulinum toxin.
 8. The complex of claim 7, whereinmember (iv) is selected from amino acids 529-562 of human SV2C (SEQ IDNO:18), amino acids 529-562 of mouse SV2C (SEQ ID NO:12), amino acids486-519 of human SV2B (SEQ ID NO:16), amino acids 486-519 of mouse SV2B(SEQ ID NO:10), and amino acids 543 to 576 of human SV2A (SEQ ID NO:14)and wherein member (viii) is selected from amino acids 454-546 of humanSV2C (SEQ ID NO:18), amino acids 454-546 of mouse SV2C (SEQ ID NO:12),amino acids 411-503 of human SV2B (SEQ ID NO:16), and amino acids468-560 of human SV2A (SEQ ID NO:14).
 9. The complex of claim 7, whereinthe polypeptide comprises a member selected from (i) amino acids 529-566of rat SV2C (SEQ ID NO:6), (ii) amino acids 486 to 523 of rat SV2B (SEQID NO:4), (iii) amino acids 543 to 580 of rat SV2A (SEQ ID NO:2), (iv) afragment of a homolog of the rat SV2C, SV2B, or SV2A wherein thefragment corresponds to amino acids 529-566 of rat SV2C, amino acids 486to 523 of rat SV2B, or amino acids 543 to 580 of rat SV2A, respectively,(v) amino acids 454-546 of rat SV2C (SEQ ID NO:6), (vi) amino acids 411to 503 of rat SV2B (SEQ ID NO:4), (vii) amino acids 468 to 560 of ratSV2A (SEQ ID NO:2), and (viii) a fragment of a homolog of the rat SV2C,SV2B, or SV2A wherein the fragment corresponds to amino acids 454-546 ofrat SV2C, amino acids 411 to 503 of rat SV2B, or amino acids 468 to 560of rat SV2A, respectively.
 10. The complex of claim 9, wherein member(iv) is selected from amino acids 529-566 of human SV2C (SEQ ID NO:18),amino acids 529-566 of mouse SV2C (SEQ ID NO:12), amino acids 486-523 ofhuman SV2B (SEQ ID NO:16), amino acids 486-523 of mouse SV2B (SEQ IDNO:10), and amino acids 543 to 580 of human SV2A (SEQ ID NO:14) andwherein member (viii) is selected from amino acids 454-546 of human SV2C(SEQ ID NO:18), amino acids 454-546 of mouse SV2C (SEQ ID NO:12), aminoacids 411-503 of human SV2B (SEQ ID NO:16), and amino acids 468-560 ofhuman SV2A (SEQ ID NO:14).
 11. The complex of claim 7, wherein theligand is BoNT/A, and wherein the polypeptide is a synthetic orrecombinant peptide.
 12. The complex of claim 9, wherein the polypeptidehas a sequence identical or homologous to a luminal domain of a murineSV2 protein.
 13. The complex of claim 9, wherein the polypeptideconsists of a member selected from (i) amino acids 529-566 of rat SV2C(SEQ ID NO:6), (ii) amino acids 486 to 523 of rat SV2B (SEQ ID NO:4),(iii) amino acids 543 to 580 of rat SV2A (SEQ ID NO:2), (iv) a fragmentof a homolog of the rat SV2C, SV2B, or SV2A wherein the fragmentcorresponds to amino acids 529-566 of rat SV2C, amino acids 486 to 523of rat SV2B, or amino acids 543 to 580 of rat SV2A, respectively, (v)amino acids 454-546 of rat SV2C (SEQ ID NO:6), (vi) amino acids 411 to503 of rat SV2B (SEQ ID NO:4), (vii) amino acids 468 to 560 of rat SV2A(SEQ ID NO:2), and (viii) a fragment of a homolog of the rat SV2C, SV2B,or SV2A wherein the fragment corresponds to amino acids 454-546 of ratSV2C, amino acids 411 to 503 of rat SV2B, or amino acids 468 to 560 ofrat SV2A, respectively.
 14. The complex of claim 13, wherein member (iv)is selected from amino acids 529-566 of human SV2C (SEQ ID NO:18), aminoacids 529-566 of mouse SV2C (SEQ ID NO:12), amino acids 486-523 of humanSV2B (SEQ ID NO:16), amino acids 486-523 of mouse SV2B (SEQ ID NO:10),and amino acids 543-580 of human SV2A (SEQ ID NO:14) and wherein member(viii) is selected from amino acids 454-546 of human SV2C (SEQ IDNO:18), amino acids 454-546 of mouse SV2C (SEQ ID NO:12), amino acids411-503 of human SV2B (SEQ ID NO:16), and amino acids 468-560 of humanSV2A (SEQ ID NO:14).
 15. The complex of claim 14, wherein thepolypeptide further consists of an affinity tag.
 16. The complex ofclaim 7, wherein the ligand is an antibody or a BoNT/A fragment thatbinds to a member of (i) to (viii) and reduces binding of BoNT/A to thepolypeptide.
 17. The complex of any one of claim 7 or claim 16, whereinthe polypeptide is a full length SV2 protein.
 18. The complex of claim16, wherein the polypeptide is located in vivo.
 19. The complex of claim7, wherein the polypeptide further comprises a binding site for aganglioside.
 20. The complex of claim 7, wherein the polypeptide is arecombinant polypeptide.
 21. The complex of claim 7, wherein thepolypeptide comprises an amino acid sequence that is at least 80%identical to any of the amino acid sequences in (i) to (viii).
 22. Amethod for reducing BoNT/A toxicity in a human or non-human animalcomprising the step of: administering to the human or non-human animalan agent that reduces binding between BoNT/A and an SV2 protein in thehuman or non-human animal wherein the agent is selected from apolypeptide comprising the polypeptide of claim 5 and an antibody ofclaim
 6. 23. The method of claim 22, wherein the method is for reducingBoNT/A toxicity in a human.
 24. The method of claim 22, wherein theagent further comprises a ganglioside.
 25. The method of claim 22,wherein the agent reduces the expression of an SV2 protein in the humanor non-human animal.
 26. The method of claim 22, wherein the agentreduces the binding between gangliosides and an SV2 protein in the humanor non-human animal.
 27. The method of claim 26, wherein the agentreduces the amount of gangliosides available for binding to the SV2protein in the human or non-human animal.
 28. A method for identifyingpolypeptide that can bind to BoNT/A comprising the steps of: providing apolypeptide that comprises a BoNT/A-binding sequence of an SV2 protein;modifying the polypeptide at the BoNT/A-binding sequence; anddetermining whether the modified polypeptide can bind to BoNT/A.
 29. Anisolated polypeptide comprising an amino acid sequenceNTYFKNCTFIDT(X1)F(X2)NTDFEPYKFIDSEF(X3)NCSF, where X1=L or V, X2=D or E,and X3=Q or K; wherein the polypeptide is capable of binding tobotulinum neurotoxin A (BoNT/A).
 30. The isolated polypeptide of claim29, wherein the polypeptide is soluble in an aqueous solvent.